Peripheral tolerance induction to alloantigen in the mature immune system remains an elusive goal. A number of maneuvers which interfere with signal 1 (antigen receptors), signal 2 (costimulatory receptors), so-called signal 3 (inflammatory cytokines), or antigen processing can result in prolonged or indefinite allograft survival via mechanisms including clonal abortion, deletion, anergy, deviation, and/or agnosia. It is not certain which methods, mechanisms, cells or molecules are most relevant for achieving nontoxic, long-lived, alloantigen-specific tolerance free from chronic immunosuppression and chronic rejection. The general failure to reach these goals suggests that there are additional levels of immune regulation. It is noteworthy that the majority of approaches to tolerance have examined molecular and cellular mechanisms in vitro, or graft survival in vivo, without regard to structural and anatomic compartmentalization that may dictate additional levels of regulation. Therefore, the definition of the anatomic domains where alloantigen is presented to induce tolerance, and where lymphocytes interact with antigen and immunosuppressants to become tolerized (deleted, anergized, deviated, ignorant) are likely to be critical determinants of the tolerization process. Our preliminary data demonstrate that T lymphocytes must utilize L-selectin to remain in the lymph node in order to induce alloantigen specific tolerance in models that perturb signal 1 or signal 2. We hypothesize that lymph node homing and localization are required for peripheral tolerance induction, and that the lymph node domain is uniquely suited to peripheral, alloantigen specific tolerance. Corollaries are that localization of T lymphocytes to other compartments, or lymph node depletion of T lymphocytes, will prevent peripheral tolerance induction. Additional implications are that quantitative distribution, such as the ratio or absolute number of T cells in lymph nodes versus other compartments (e.g., peripheral blood, spleen), and temporal distribution during tolerization regimens determine the balance between immunity and tolerance. The Specific Aims will investigate this hypothesis as follows: 1) Determine why inhibition of CD62L dependent T cell LN homing prevents tolerance. 2) Determine what other receptors and ligands are important for T cell homing and tolerance induction. 3) Determine the role of specific anatomic sites during tolerance induction.